Acquisition and management of metal ions is a critical part of metabolism for all forms of life because metalloproteins comprise close to one third of all proteins and almost half of all enzymes. Metals must be handled such that the correct ions are provided to essential enzymes and proteins, but do not accumulate to deleterious levels. A host of proteins, including membrane transporters, metallochaperones, and metal sensors, maintain metal ion concentrations in cells and cellular compartments. Aberrant handling of metal ions such as copper, zinc, iron, and manganese is linked to numerous human diseases. In addition, a number of bacterial metal trafficking proteins have been identified as virulence factors. The long term objective of this research program is to understand metal homeostasis on the molecular level. The P1B-type ATPases, integral membrane proteins that use the energy of ATP hydrolysis to transport metal ions across membranes, play a key role in metal homeostasis in all organisms. Distinct subfamilies of P1B-ATPases transport different transition metal ions, including Zn2+/Cd2+/Pb2+, Cu2+, Cu+/Ag+, and Co2+. The P1B-1-ATPase subclass includes the human Wilson and Menkes disease proteins, mutations in which lead to disorders of copper metabolism and which are implicated in resistance to anticancer drugs. Despite their universal importance, very little is known about the biochemical, structural, and metal binding properties of the different P1B-ATPase subfamilies. In particular, the molecular basis for metal ion specificity remains unclear. The proposed research involves biochemical and biophysical studies of selected P1B-ATPases, which have been chosen on the basis of sequence and architectural considerations as well as experimental tractability. The approach includes cloning, overexpression, protein purification, metal binding studies, in vitro activity assays, in vivo assays, site-directed mutagenesis, spectroscopy, and crystallography. The first two specific aims focus on characterization of Cu+ transporting CopA P1B-1-ATPases, the soluble metal binding domains of the Cu2+ specific P1B-3-ATPases, and the putative Co2+ transporting P1B-4-ATPases. The third specific aim focuses on identifying the metal substrate of the P1B-5-ATPase subfamily, of which some members contain a novel C-terminal hemerythrin-like domain, the function of which is not known. The last specific aim is crystallization and structure determination of intact P1B-ATPases using several innovative approaches. Taken together, this body of work will address what is currently a huge gap in understanding of metal homeostasis. PUBLIC HEALTH RELEVANCE: A number of human diseases are linked to deficiencies in handling of essential yet potentially toxic metal ions. In addition, human metal transporting proteins are associated with resistance to anticancer drugs and the virulence of human pathogens is dependent on metal acquisition. This project will provide a molecular level understanding of how human and bacterial proteins transport metal ions across cell membranes.